Effects of Bothrops asper snake venom on lymphatic vessels: insights into a hidden aspect of envenomation.

Mora J, Mora R, Lomonte B, Gutiérrez JM - PLoS Negl Trop Dis (2008)

Bottom Line:
B. asper venom induced a dose-dependent contraction of collecting lymphatic vessels, resulting in a reduction of their lumen and in a halting of lymph flow.In agreement with this, treatment of the venom with fucoidan, a myotoxin inhibitor, abrogated the effect, whereas no inhibition was observed after incubation with the peptidomimetic metalloproteinase inhibitor Batimastat.Moreover, fucoidan significantly reduced venom-induced footpad edema.

Background: Envenomations by the snake Bothrops asper represent a serious medical problem in Central America and parts of South America. These envenomations concur with drastic local tissue pathology, including a prominent edema. Since lymph flow plays a role in the maintenance of tissue fluid balance, the effect of B. asper venom on collecting lymphatic vessels was studied.

Methodology/principal findings: B. asper venom was applied to mouse mesentery, and the effects were studied using an intravital microscopy methodology coupled with an image analysis program. B. asper venom induced a dose-dependent contraction of collecting lymphatic vessels, resulting in a reduction of their lumen and in a halting of lymph flow. The effect was reproduced by a myotoxic phospholipase A(2) (PLA(2)) homologue isolated from this venom, but not by a hemorrhagic metalloproteinase or a coagulant thrombin-like serine proteinase. In agreement with this, treatment of the venom with fucoidan, a myotoxin inhibitor, abrogated the effect, whereas no inhibition was observed after incubation with the peptidomimetic metalloproteinase inhibitor Batimastat. Moreover, fucoidan significantly reduced venom-induced footpad edema. The myotoxic PLA(2) homologue, known to induce skeletal muscle necrosis, was able to induce cytotoxicity in smooth muscle cells in culture and to promote an increment in the permeability to propidium iodide in these cells.

Conclusions/significance: Our observations indicate that B. asper venom affects collecting lymphatic vessels through the action of myotoxic PLA(2)s on the smooth muscle of these vessels, inducing cell contraction and irreversible cell damage. This activity may play an important role in the pathogenesis of the pronounced local edema characteristic of viperid snakebite envenomation, as well as in the systemic biodistribution of the venom, thus representing a potential therapeutical target in these envenomations.

pntd-0000318-g009: Inhibition of the cytotoxic activity of B. asper venom on smooth muscle cells in culture.Venom was incubated with fucoidan for 60 min at room temperature, and cytotoxicity was tested as described in Methods and in the legend of Fig 7. Controls included PBS alone, venom incubated with PBS, and fucoidan alone. The dose-dependent cytotoxic effect of venom, reflected in LDH release, was completely abrogated by incubation with fucoidan. Results are presented as mean±S.D. (n = 3). * p<0.05 when compared with cells incubated with venom+fucoidan.

Mentions:
In order to gain insights into the mechanism through which B. asper venom affects lymphatics, cultured smooth muscle cells were treated with myotoxin II. Myotoxin induced a rapid and dose-dependent cytotoxicity, evidenced by the release of the cytosolic enzyme LDH (Fig 7). The toxin also induced a rapid increment in the permeability of plasma membrane to PI (Fig 8) which corroborated the disruption in the integrity of plasma membrane. Moreover, incubation of B. asper venom with fucoidan, prior to its addition to smooth muscle cell culture completely abrogated cytotoxicity in these cells (Fig 9), thus confirming the relevance of basic myotoxins in this effect.

pntd-0000318-g009: Inhibition of the cytotoxic activity of B. asper venom on smooth muscle cells in culture.Venom was incubated with fucoidan for 60 min at room temperature, and cytotoxicity was tested as described in Methods and in the legend of Fig 7. Controls included PBS alone, venom incubated with PBS, and fucoidan alone. The dose-dependent cytotoxic effect of venom, reflected in LDH release, was completely abrogated by incubation with fucoidan. Results are presented as mean±S.D. (n = 3). * p<0.05 when compared with cells incubated with venom+fucoidan.

Mentions:
In order to gain insights into the mechanism through which B. asper venom affects lymphatics, cultured smooth muscle cells were treated with myotoxin II. Myotoxin induced a rapid and dose-dependent cytotoxicity, evidenced by the release of the cytosolic enzyme LDH (Fig 7). The toxin also induced a rapid increment in the permeability of plasma membrane to PI (Fig 8) which corroborated the disruption in the integrity of plasma membrane. Moreover, incubation of B. asper venom with fucoidan, prior to its addition to smooth muscle cell culture completely abrogated cytotoxicity in these cells (Fig 9), thus confirming the relevance of basic myotoxins in this effect.

Bottom Line:
B. asper venom induced a dose-dependent contraction of collecting lymphatic vessels, resulting in a reduction of their lumen and in a halting of lymph flow.In agreement with this, treatment of the venom with fucoidan, a myotoxin inhibitor, abrogated the effect, whereas no inhibition was observed after incubation with the peptidomimetic metalloproteinase inhibitor Batimastat.Moreover, fucoidan significantly reduced venom-induced footpad edema.

Background: Envenomations by the snake Bothrops asper represent a serious medical problem in Central America and parts of South America. These envenomations concur with drastic local tissue pathology, including a prominent edema. Since lymph flow plays a role in the maintenance of tissue fluid balance, the effect of B. asper venom on collecting lymphatic vessels was studied.

Methodology/principal findings: B. asper venom was applied to mouse mesentery, and the effects were studied using an intravital microscopy methodology coupled with an image analysis program. B. asper venom induced a dose-dependent contraction of collecting lymphatic vessels, resulting in a reduction of their lumen and in a halting of lymph flow. The effect was reproduced by a myotoxic phospholipase A(2) (PLA(2)) homologue isolated from this venom, but not by a hemorrhagic metalloproteinase or a coagulant thrombin-like serine proteinase. In agreement with this, treatment of the venom with fucoidan, a myotoxin inhibitor, abrogated the effect, whereas no inhibition was observed after incubation with the peptidomimetic metalloproteinase inhibitor Batimastat. Moreover, fucoidan significantly reduced venom-induced footpad edema. The myotoxic PLA(2) homologue, known to induce skeletal muscle necrosis, was able to induce cytotoxicity in smooth muscle cells in culture and to promote an increment in the permeability to propidium iodide in these cells.

Conclusions/significance: Our observations indicate that B. asper venom affects collecting lymphatic vessels through the action of myotoxic PLA(2)s on the smooth muscle of these vessels, inducing cell contraction and irreversible cell damage. This activity may play an important role in the pathogenesis of the pronounced local edema characteristic of viperid snakebite envenomation, as well as in the systemic biodistribution of the venom, thus representing a potential therapeutical target in these envenomations.